Lactobacillus sake like strains, production and use of their exopolysaccharides

ABSTRACT

New Lactobacillus sake like strains are provided obtainable from meat products, which strains are capable of producing an exopolysaccharide having shear-thinning properties, even at low concentrations, and/or thickening and/or emulsion-stabilizing properties.

This is a 35 U.S.C. 371 national application of PCT/EP93,03338, filedNov. 26, 1993.

BACKGROUND OF THE INVENTION

A large number of food products like mayonnaises, dressings, margarines,spreads or low-fat or zero-fat substitutes, can be stabilized bypolysaccharides as emulsion stabilizers or thickening agents. Also inthe medical, pharmaceutical and cosmetic fields, polysaccharides areused as emulsion stabilizers. Well known polysaccharides are obtainedfrom a variety of plant seeds, e.g. guar gum from Cyamopsistetragonaloba (guar), or locust bean gum (LBG) from locust bean. Otherwell known sources are seaweeds, giving carrageenan, alginates or agar.

One disadvantage of all these polysaccharides is, that the supply of thesources and thus the constant availability of the polysaccharides, isnot certain, while demands are ever growing. This has already led tohigh and fluctuating prices for a highly functional polysaccharide likeLBG. As an option to produce a product at reasonable prices the processdescribed in

EP-B-0121960 (UNILEVER) was developed for converting the cheaper, butless versatile polysaccharide guar gum into a clearly improved gum. Apotentially commercial production of the required guar α-galactosidasewas described in WO-A-87/07641 (UNILEVER)=U.S. Pat. No. 5,082,778.

Another disadvantage is that the isolation procedure for polysaccharidesfrom seeds or seaweed is rather cumbersome.

A further disadvantage is that most of the polysaccharides have not thehighly wanted, non-Newtonian property of shear-thinning, which is theeffect that the viscosity reversibly decreases as the shear is enhanced,even at low concentrations.

A polysaccharide that both has the wanted shear-thinning property and isreliable with respect to production and isolation, is xanthan gum, apolysaccharide produced by the bacterium Xanthomonas campestris throughfermentation. Consequently, xanthan gum is increasingly used in food andmedical products and is expected to have by far the highest growthpotential in the coming years. However, the producing microorganism,Xanthomonas campestris, is not food-grade. Moreover, a food productcontaining added xanthan gum has often to be labelled as containing anadditive. This is disadvantageous in view of the present trends towardshealthy "green" or only scarcely labelled food products.

Thus a need exists for a polysaccharide produced by a food-grademicroorganism, having properties similar to or even superior to xanthangum. Such a polysaccharide can either be added to the food product andthe resulting product has to be labelled (but then the product is aso-called "friendly labelled" additive), or it can be produced in situwithout the necessity of any labelling, because the microorganism isfood-grade.

SUMMARY OF THE INVENTION

The present invention is based on the results of a large screeningprogram involving about 600 lactic acid bacteria strains present invarious food products, e.g. olives, traditional cheeses, and sour dough,which screening program resulted in the isolation of 30exopolysaccharides-producing strains having thickening and/oremulsion-stabilizing properties. However, only some of these 30lactobacilli, which appeared to be present in meat products, especiallyin Belgian sausages, produce exopolysaccharides (EPS) having (1)thickening and/or (2) emulsion stabilizing properties and/or even show(3) the highly desirable shear-thinning property, in particular at lowconcentrations. As an example thereof a Lactobacillus strain wasisolated, characterised as a Lactobacillus sake like strain and giventhe name Lactobacillus sake like strain O-1. This strain, combining thethree different properties mentioned above, was deposited under theconditions of the Budapest Treaty at the Centraalbureau voorSchimmelcultures in Baarn, The Netherlands with number CBS 532.92.

The present invention relates to an EPS obtainable from saidLactobacillus sake like strain O-1 as well as to exopolysaccharidesobtainable from similar Lactobacillus sake like strains having eitherviscosity-increasing properties, or shear-thinning properties, or both.The EPS obtainable from Lactobacillus sake like strain O-1 comprisesunits of the monosaccharides rhamnose and glucose.

The invention also relates to a Lactobacillus sake like strain capableof producing such EPS. A preferred strain is the Lactobacillus sake likestrain O-1, deposited as CBS 532.92.

The invention further relates to a process for producing such EPScomprising (a) growing a Lactobacillus sake like strain capable offorming an EPS in a suitable medium under conditions whereby said EPS isformed, and optionally (b) isolating the EPS formed. In one embodimentof this aspect of the invention such process can be used for theproduction of an EPS or an EPS-containing product, either of which canbe used as an additive for food products or in medical, pharmaceuticaland cosmetic applications. In another embodiment the process can be usedfor in situ production of an EPS comprising growing a Lactobacillus sakelike strain in a dairy liquid medium under conditions whereby said EPSis formed until the culture has a relative high density of saidlactobacilli. Preferably the product obtained by said process is notthereafter subjected to an intensive shear treatment. Such culturecontaining an EPS can advantageously be incorporated into dairyingredient-containing products such as dressings, margarine, mayonnaise,and spreads, and low-fat or zero-fat substitutes therefor.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1 the UPGMA (Unweighted Pair Group Method using Average linkage)dendrogram of the Pearson Product Moment Correlation coefficients(r×100) between SDS-PAGE protein patterns is given. The strains assignedwith a "LAB"-number are stored in the closed "Lactic Acid Bacteria"culture collection of the University of Ghent. Strains assigned with a"LMG"-number belong to the "Laboratorium voor Microbiologie, Gent"culture collections. The reference strains used in this study areindicated in the dendrogram, as are the EPS-producing strains. Thestrain "LAB28" (O-1) refers to the Lactobacillus sake like strain O-1with deposit number CBS 532.92.

In FIG. 2 the shear rate versus viscosity and shear rate versus shearstress of a 1% (wt./vol.) aqueous solution of both the EPS fromLactobacillus sake like strain O-1 and xanthan gum are plotted.

shear stress 1% 0-1 EPS viscosity 1% O-1 EPS

shear stress 1% xanthan viscosity 1% xanthan

In FIG. 3 the structure of a preferred EPS according to the invention isprovided.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect the invention relates to a polysaccharide that can beproduced by a Lactobacillus sake like strain, isolated from a meatproduct, e.g. a Belgian sausage. Thus the invention provides apolysaccharide obtainable from a food-grade Lactobacillus sake likestrain. One preference is for a polysaccharide comprising units of themonosaccharides rhamnose and glucose, more preferably in a ratio ofabout 1:4 to about 1:1 most preferably in a ratio of 3:7 to 1:1.

It has been found that the EPS obtainable from Lactobacillus sakegenerally comprises acetyl and phospho glycerol side groups. The EPS isbelieved to be partially O-acylated, whereby the level of acylation isfrom 0.40 to 0.45 per rhamnose unit for example 0.425 per rhamnose unit.The level of phosphorylation is believed to be more than 0.45 perrhamnose unit, for example 0.50 per rhamnose unit. FIG. 3 gives the mostlikely structure as obtained by NMR techniques of the EPS obtained fromLactobacillus sake O-1. The EPS is composed of repeating pentasaccharideunits with 3 glucose units and 2 rhamnose units, whereby one of therhamnose units has a 1-phospho glycerol substituent and the otherrhamnose unit contains on average about 0.85 O-acetyl groups.

For some embodiments of the invention it may be useful to reduce thenumber of substituent groups. This may be accomplished by conventionalchemical techniques, for example the EPS may be O-deacylated by mildalkalic treatment, for example with ammonia. Also the EPS may besubjected to a highly alkaline treatment (NaOH) for furtherO-deacetylation and dephosphorylation. It is believed that removal ofthe side-groups may influence the rheology properties of the EPS.

Another preference is that the polysaccharide according to the inventiongives a viscosity of at least 10 mPa.s when measured with a HaakeRotovisco RV100 viscosimeter at a concentration of 2 g/l (=0.2% wt.) inwater and a shear rate of 300 s⁻¹, or a viscosity of at least 1000 Mpa.swhen measured at a concentration of 10 g/l (=1% wt.) in water and ashear rate of 3 s⁻¹. Especially preferred is a viscosity of more than100 Pa.s at a shear rate of 0.01 s⁻¹ and a concentration of 1 wt % inwater.

Other preferred rheology characteristics of the EPS of the inventionare: a value p of between 0.7 and 0.9, wherein the viscosity(Pa.s)≈(shear rate (s⁻¹)^(-p). Other characteristics are: viscosity of 1wt % solution at 0.01 s⁻¹ is relatively T-independent (less than 50%fluctuation in the range 4° to 20° C.), pH independent (less than 10%fluctuation in pH range 4-6) and relatively independent on the presenceof salts. Furthermore it has been observed that a synergistic increasein viscosity can be observed if the EPS of the invention is combinedwith guar gum or LBG. Also the EPS of the invention tend to act as athickener rather than a gelling agent. This difference can be shown byshear modulus measurements.

Another preferred feature of a polysaccharide according to the inventionis, that it has shear-thinning properties in that the viscosity isreversibly reduced upon increasing the shear rate. A most preferredpolysaccharide is one that is obtainable from Lactobacillus sake likestrain O-1 deposited as CBS 532.92. This latter polysaccharide has thefollowing properties:

External appearance: tasteless and odourless white powder;

Solubility: readily soluble in water, scarcely soluble in methanol,ethanol and acetone;

Composition: 60-65% glucose units and 35-40% rhamnose units.

Viscosity: measured in an aqueous solution at 25° C. for both EPS fromLactobacillus sake like strain O-1 and xanthan as comparison

    ______________________________________                                                          viscosity in mPa.s                                          concentration                                                                            shear rate   O-1 EPS  xanthan                                      ______________________________________                                        0.2% wt.   300 s.sup.-1  18       14                                          1.0% wt.    3 s.sup.-1  2440     1240                                         ______________________________________                                    

In another aspect the invention provides a process for producing apolysaccharide according to the invention, which process comprises (a)growing a Lactobacillus sake like strain capable of forming an EPS in asuitable medium under conditions whereby said EPS is formed, andoptionally (b) isolating the EPS formed. A suitable medium appeared tobe a so-called Semi Defined Medium (SDM) consisting of a carbon sourcelike glucose or another fermentable carbohydrate, at least one nitrogensource, a phosphate source, e.g. a mixture of Na₂ HPO₄ and KH₂ PO₄ insuch a concentration that it also has a high buffering capacity,vitamins, minerals (especially Mn²⁺), amino acids and peptide mixtures.Two SDM's are given in Examples 1 and 6. As will be apparent to askilled person these SDM's for laboratory trials need to be adapted whencommercially producing the EPS at a larger scale. The strain isgenerally grown between 15° C. and 40° C. for 24 to 48 hours, withoutaeration for optimal production of the EPS. The EPS can be isolated fromthe fermentation broth by any suitable technique e.g. by means of aprecipitation process using an organic solvent in which the EPS is notsoluble or has a limited solubility. Another possibility is removal ofthe water, e.g. by evaporation, as is often done in the production ofxanthan gum on a commercial scale, or by membrane filtration.

The thus isolated polysaccharide can be applied as such as an additiveto food products. For convenience and easy handling, it is howeversometimes preferred to apply the polysaccharide to a carrier material.This can be accomplished by any suitable technique. Any edible carriermaterial can be used, although the use of protein carriers e.g. wheyprotein or soy protein is especially preferred.

The polysaccharide can be applied as a "friendly" labelled additive inthe production of dressings, mayonnaise, spreads and their low-fat andzero-fat equivalents, creams, sauces, meat (products) cheese, puddings,ice-cream and bakery products, using processes familiar to those skilledin the art, which will be further illustrated in the Examples. The levelof polysaccharide will generally be from 0.01 to 15 wt %, more preferred0.1 to 10 wt %, most preferred 0.5 to 5 wt %.

For some of these applications (e.g. cheese) it is preferred that theproduct is prepared under relatively quiescent conditions, e.g. gentleor no stirring after mixing the ingredients in order to avoid undesiredshear-thinning.

Thus the invention also provides a process for producing a food productcontaining a thickened aqueous phase, which process comprisesincorporating into said food product an effective amount of apolysaccharide according to the invention.

Another embodiment of this aspect of the invention which does notrequire labelling the food product as containing an additive, is aprocess in which the EPS is formed by the Lactobacillus sake like strainduring fermentation of a commercially acceptable medium for startercultures, after which the water can be removed by conventionaltechniques, e.g. evaporation, membrane filtration, or spray-drying.

Labelling is also not required when the polysaccharide is produced insitu by a food-grade micro-organism. An example is a process forproducing a mayonnaise type product, in which milk or a milk-basedmedium, e.g. pasteurised skimmilk containing Hysoy (ex QuestBioproducts, U.S.A.) and MnSO₄, is fermented with the Lactobacillus sakelike strain at 30° C. for 18 hours until a sufficient high amount oflactobacilli is formed producing sufficient EPS. Subsequently thefermented milk product is carefully mixed both with an aqueous phasecontaining usual ingredients, e.g. salt, sugar, acids, flavourcomponents and starch, and with a separately prepared mayonnaisepre-emulsion phase.

Thus the invention also provides a process for in situ producing apolysaccharide according to the invention, comprising growing aLactobacillus sake like strain in an edible medium e.g. a dairy liquidmedium under conditions whereby said EPS is formed until the number ofsaid lactobacilli is in the order of 10⁷ to 10¹¹ per ml, preferably 10⁹to 10¹⁰ per ml. Preferably the product obtained by said process is notsubsequently subjected to an intensive shear treatment.

Another embodiment of this aspect of the invention is a process forproducing a product containing a dairy ingredient such as dressings,margarine, mayonnaise, and spreads, and low-fat or zero-fat substitutestherefor, which comprises incorporating into said product a liquid dairymedium obtained by a process for in situ producing a polysaccharideaccording to the invention. The dairy ingredient can form the totalaqueous phase or at least part of the aqueous phase of said product.

The invention is further illustrated by the following Examples, which donot limit the scope of the present invention. Percentage in thefollowing Examples are expressed as wt. unless otherwise stated.

EXAMPLE 1 Search for exopolysaccharide-producing lactic acid bacteriapresent in sausages

From 5 different home-made, traditionally fermented, chourico sausagesfrom the region of Alenteijo in Portugal and from a traditionallyfermented salami sausage from Recogne in Belgium, 10 g of sausage wassuspended in 90 ml of 0.85% NaCl solution using a stomacher model BA6021(ex Seward Laboratory). These suspensions were diluted 10⁻², 10⁻⁴ and10⁻⁶ times in 0.85% NaCl and 0.1 ml of the respective dilutions wasplated out on MRS-agar which contained 20 mg pimafucine (ex Duchefa) per100 ml medium to prevent growth of yeasts or fungi. The plates wereincubated anaerobically at 30° C. for 48 hours.

In total 159 lactic acid bacteria strains were isolated. All strainswere selected for the production of EPS as follows. The individualstrain was grown overnight in MRS-broth at 30° C. "EPS selection medium"(ESM), composed of 9% skimmilk powder (ex Frico Domo), 0.35% YeastExtract (ex Difco), 0.35% Bacto Pepton (ex Difco) and 1% glucose wasinoculated with 1% (v/v) of this overnight culture. After 24 hoursincubation at 30° C., the milk-curd was checked for "ropiness". To thisend 1 ml of the milk-curd was taken up in a 1 ml volumetric pipet. Thepipet was emptied again and when drops falling from the tip of the pipetshowed a slimy behaviour, this referred to EPS-production in the ESM.With this method 4 of the 159 lactic acid bacteria strains isolatedturned out to be EPS producers. These 4 strains were all isolated fromthe Belgium salami, whereas no EPS-producing strains could be isolatedfrom the Portuguese sausages investigated.

The viscosity produced by a 1% (v/v) inoculum of an overnight grownculture (in MRS broth) in a Semi Defined Medium (SDM-1) (containing 2.5g K₂ HPO₄, 3.0 g KH₂ PO₄, 0.6 g (NH₄)₂ -citrate, 1.0 g Na-acetate, 5.0 gcasamino acids (ex Difco), 6.7 g Yeast Nitrogen Base (ex Difco) and as acarbon source 2% glucose) incubated 24 hours at 30° C. was measured withthe Haake Rotovisco RV100; system CV100 (sensor system ZA-30) at a shearrate of 300 s⁻¹. The viscosity of the 4 strains isolated from theBelgium salami turned out to be approximately the same and ranged from1.9 to 2.2 mPa.s. As a typical strain we have chosen the one showing thehighest viscosity, produced when grown under the above mentionedconditions.

This strain was named Lactobacillus sake like strain O-1. In thisspecification the abbreviated forms "strain O-1" and "O-1" are alsoused.

EXAMPLE 2 Physiological and taxonomical analysis of theexopolysaccharide producing Lactobacillus sake like strain O-1

The investigated strain O-1 is gram-positive, catalase-negative,immotile, and has rod-shaped bacteria occurring in short chains of inbetween 1 to 5 cells when grown in MRS broth. This strain was able toferment the following sugar substrates (determined with API 50 CHL):L-arabinose, ribose, galactose, D-glucose, D-fructose, D-mannose,N-acetyl glucosamine, esculin, salicin, cellobiose, lactose, andsaccharose.

The taxonomical identification of the lactic acid bacteria isolated fromthe various fermented sausages as mentioned in Example 1 was carried outusing polyacrylamide gel-electrophoresis of proteins. The strains weregrown on MRS agar in Roux flasks at 30° C. for 24 hours. Roux flaskswere inoculated from a 24 hours grown MRS broth culture. Whole cellprotein extracts were prepared as described previously (Kiredjan, 1986).Cells were lysed by sonication using a Labsonic 2000 sonicator (ex B.Braun, Melsungen, Germany) with a needle probe tip (length 127 mm,diameter 4 mm) during 3 minutes in position "LOW" with 50 W output.Sodium dodecylsulphate polyacrylamide gel-electrophoresis was carriedout using the procedure of Laemmli (1970), which was modified asdescribed by Kiredjan (1986). Registration of the proteinelectrophoretic patterns, normalization of the densitometric traces,grouping of strains by the Pearson product moment correlationcoefficient (r) and UPGMA (Unweighted Pair Group Method using Averagelinkage) cluster analysis were performed by the techniques described byPot (1992), using the software package GELCOMPAR (version 2.0; L.Vauterin & P. Vauterin, commercially available at Applied Maths,Risquons-Toutstraat 38, B-8511 Kortrijk, Belgium).

This method was used for the identification of 32 strains that wereisolated from the traditionally fermented Belgium salami as mentioned inExample 1. Of these 32 strains 4 were able to produce EPS, one of whichwas the above indicated strain O-1. For the identification of these 32strains the protein patterns were compared with the protein patterns of600 lactic acid bacteria reference strains which were stored in adatabase. In FIG. 1 the UPGMA dendrogram of the mean correlationcoefficient (r), calculated between all pairs of strains, is presented.Both the investigated strains and the closely related reference strainsare indicated, clearly showing that the strains from the Belgium salamioccupied a taxonomic position separate from the Lactobacillus sakereference strains. However, three reference strains which werephenotypically identified as Lactobacillus sake but showed aberrantprotein patterns, were closely related to the strains isolated from theBelgian salami. These reference strains were known as "Lactobacillussake like" and therefore the strains isolated from the Belgian salamiwere also considered to be "Lactobacillus sake like".

EXAMPLE 3 Isolation and purification of the exopolysaccharide producedby the Lactobacillus sake like strain O-1

A single colony of strain O-1 was selected from a MRS agar plate,transferred to fresh MRS broth and incubated overnight at 30° C. Freshlyprepared Semi Defined Medium (SDM-1), as described in Example 1, wasinoculated with 2.5% (v/v) of the overnight grown strain O-1 culture.This inoculated SDM-1 was incubated for 24 hours at 30° C. withoutaeration. To remove proteins from the culture broth trichloroacetic acid(TCA) was added until a concentration of 4% was reached. After gentlemixing the culture was allowed to stand for 30 minutes at roomtemperature. The culture was centrifuged for 30 minutes at 27000 g andthe clear supernatant was collected. The EPS produced was precipitatedwith 2.5 volumes of cold ethanol. The precipitate was collected,redissolved in approximately 5% (v/v) of the original volume of waterand dialysed against demineralized water for 2 days at 4° C. The waterwas refreshed twice a day. After dialysis a fractionated acetoneprecipitation was carried out. The bulk of the dissolved EPS wasprecipitated in the 50% (v/v) acetone fraction. This fraction containedabout 80 wt.-% of the total amount of EPS and was 99 wt.-% pure, i.e.protein contamination<1 wt.-%. When an ≈100 wt.-% pure EPS is requiredthe material can be further purified by gel filtration on a column ofSephacryl S-500 (ex Pharmacia). The precipitate of this Example wasredissolved in demineralized water and lyophilized. The material wasstored under dry conditions at 4° C.

EXAMPLE 4 Compositional analysis of the exopolysaccharide produced bythe Lactobacillus sake like strain O-1

The sugar composition of the EPS produced by strain O-1 was determined.After methanolysis, followed by N-(re)acetylation andtrimethylsilylation of the EPS the methyl glycosides of the differenttypes of monosaccharides were determined by GC-MS. The EPS was composedof 60-70 wt.-% glucose and 30-40 wt.-% rhamnose.

EXAMPLE 5 Rheological behaviour of both the exopolysaccharide producedby the Lactobacillus sake like strain O-1 and xanthan gum produced byXanthomonas campestris

The viscosity of a 1% aqueous solution of the EPS produced by strain O-1was determined as a function of the shear rate. All rheologicalmeasurements were carried out at 25° C. using a Haake Rotovisco RV100;system CV100; sensor system ZA-30. In FIG. 2 the viscosity profile iscompared with that of a 1% aqueous solution of xanthan gum (ex Kelco).In FIG. 2 it is clearly shown that the useful shear-thinning property ofxanthan gum is also present in the O-1 EPS.

Detailed measurements at a shear rate of 3 s⁻¹, showed that theviscosity of a 1% aqueous solution of the O-1 EPS was 2440 mPa.s, wherethe viscosity of a 1% aqueous solution of xanthan gum was 1240 mPa.s.The viscosities measured at a shear rate of 300 s⁻¹ of a 1% aqueoussolution of O-1 EPS and of xanthan gum were determined as 70 and 54mPa.s respectively, whereas the viscosities measured at a shear rate of300 s⁻¹ of a 0.2% aqueous solution of O-1 EPS and of xanthan gum weredetermined as 18 and 14 mPa.s, respectively.

EXAMPLE 6 Optimization of the growth of the exopolysaccharide producingLactobacillus sake like strain O-1

In order to study the physiology of the production of EPS by strain O-1a defined growth medium was desired. Since MRS is a rich, non-specifiedmedium (containing yeast extract, beef extract etc.) and the medium usedin Example 1 resulted in a poor growth (optical density at 610 nm was≈1.2), a new Semi Defined Medium (SDM-2) was developed. For 1 literSDM-2 the following components were weighed out: 10.0 g K₂ HPO₄, 12.4 gNaH₂ PO₄, 0.6 g (NH₄)₂ -citrate, 0.05 g MnSO₄, 5.0 g casamino acids (exDifco), 10.0 g Bacto proteose pepton (ex Difco), 6.7 g Yeast NitrogenBase (ex Difco) and 20 g glucose. The final pH of the medium was 6.5.After incubation of strain O-1 in this SDM-2 at 35° C. in non-shakenErlenmeyer flasks without pH control the OD₆₁₀ was 5.0, whereas growthin MRS resulted in a OD₆₁₀ of 3.5-4.0. Anaerobic incubation in SDM-2 at20° C. for 48 hours resulted in a OD₆₁₀ of 6.5, whereas the viscosity ofthe culture broth was 3.8 mPA.s compared with a highest value of 2.2when grown in SDM-1 (see Example 1). The concentration of the EPS inbroth was 90 mg/l compared with a value of 30 mg/l when grown in SDM-1.

EXAMPLE 7 In situ production of the exopolysaccharide of Lactobacillussake like strain O-1 in a milk-based medium

300 g milk powder and 30 g Hysoy (ex Quest Bioproducts, USA) weredissolved in water, up to an end volume of 3 l. It was pasteurised byincubating it for 1 minute at 90° C. Subsequently, 0.3 mM MnSO₄ wasadded and after cooling to about 30° C., the milk medium was inoculatedwith 1% (v/v) of an overnight culture of Lactobacillus sake like strainO-1, grown in the same medium. After incubating the culture for 18 hoursat 30° C., a highly viscous fermented milk was obtained, which was usedin the production of a low-fat dressing, as shown in the followingExample.

EXAMPLE 8 Preparation of a dressing based on the in situ producedexopolysaccharide of Lactobacillus sake like strain O-1

A pre-emulsion phase was made by mixing equal volumes of vinegar and amixture of water, egg-yolk, edible oil and B-carotene at low shear. Thispre-emulsion phase was mixed with an aqueous phase containing sorbate,sugar, salt, vinegar, citric acid and instant starch and subsequentlythe mixture was homogenised at high shear. The exact composition of themixtures depends on the properties, required for the final product. Tothis emulsion, the viscous fermented milk, the preparation of which wasdescribed in the preceding Example, was added at an end concentration of10-20% (v/v). The mixture was carefully homogenised under low shear. Adressing having excellent body and taste was obtained.

EXAMPLE 9

A spread can be obtained by preparing an aqueous phase containing 2 wt %(on product) of the exopolysaccharide as described above and mixing 60parts of the aqueous phase with 40 parts of a fat phase containingbutterfat and 1% (on product) of monoglycerides.

EXAMPLE 10 Improved production of EPS

The following medium was used

    ______________________________________                                        Na.sub.2 HPO.sub.4 10         g/liter                                         KH.sub.2 PO.sub.4  12         g/liter                                         (NH.sub.4).sub.2 -citrate                                                                        0.6        g/liter                                         MnSO.sub.4         0.05       g/liter                                         NZ-case Plus (exQuest)                                                                           20         g/liter                                         glucose            20         g/liter                                         Yeast nitrogen base (ex Difio)                                                                   6.7        g/liter                                         pH (NaOH)          5.8                                                        water              balance                                                    ______________________________________                                    

Lactobacillus sake O-1 was anaerobically grown on this medium at 20° C.while maintaining the pH at 5.8 for about 36 hours. Under theseconditions 1350 mg EPS/l is produced. The structure of the EPS isdetermined by NMR and is as represented in FIG. 3 (ratio glucose:rhamnose 3:2, 0.425 O-acetyl groups per rhamnose unit, 0.5 1-phosphoglycerol group rhamnose unit).

We claim:
 1. Food grade exopolysaccharide obtainable from a food grademicroorganism, said exopolysaccharide consisting of monosaccharide unitsrhamnose and glucose in the ratio of about 1:4-1:1 and possessing shearthinning properties in that the viscosity is reversibly reduced uponincreasing the shear rates and possessing at least one of the propertiesof thickening and emulsion stabilizing.
 2. Food grade exopolysaccharideaccording to claim 1 wherein rhamnose and glucose are in the ratio of3:7-1:1.
 3. Food grade exopolysaccharide according to claim 1 possessingrepeating pentasaccharide units with 3 glucose and 2 rhamnose.
 4. Foodgrade exopolysaccharide according to claim 1 possessing 60-65% glucoseand 35-40% rhamnose.
 5. Food grade exopolysaccharide according to claim1, wherein said at least one property is thickening property.
 6. Foodgrade exopolysaccharide according to claim 1, wherein said at least oneproperty is emulsion stabilizing property.
 7. Food gradeexopolysaccharide according to claim 1 which possesses acetyl andphosphoglycerol side groups, being O-acylated at the rhamnose unit andphosphorylated at the rhamnose unit.
 8. Food grade exopolysaccharideaccording to claim 1 wherein rhamnose is phosphorylated with a1-phosphoglycerol substituent.
 9. Food grade exopolysaccharide accordingto claim 1 with a viscosity of at least 10 mPA.s when measured with aviscosimeter in a concentration of 0.2 wt % in water and a shear rate of300 s⁻¹ or of at least 1000 mPa.s when measured in concentration of 1 wt% in water and a shear rate of 3 s⁻¹ or of at least 100 Pa.s whenmeasured in a concentration of 1 wt % in water at a shear rate of 0.01s⁻¹.
 10. Food grade exopolysaccharide according to claim 1 obtained froma microorganism strain with the phenotypical characteristics ofLactobacillus sake with aberrant protein patterns determinable from theUPGMA dendogram of the mean correlation coefficient (r).
 11. Food gradeexopolysaccharide according to claim 1 obtainable from Lactobacillussake-like strain O-1 deposited at the Centraal Bureau voorSchimmelcultures in Baarn, the Netherlands under accession number CBS532.92.
 12. A Lactobacillus sake-like strain capable of producing anexopolysaccharide according to claim 1, said strain having thephenotypical characteristics of Lactobacillus sake with aberrant proteinpatterns determinable from the UPGMA dendogram of the mean correlationcoefficient (r).
 13. Lactobacillus sake-like strain according to claim12, said strain being deposited at the Centraal Bureau voorSchimmelcultures in Baarn, the Netherlands under accession number532.92.
 14. A method for producing an exopolysaccharide according toclaim 1 comprising growing a Lactobacillus sake-like strain in asuitable medium under conditions whereby the exopolysaccharide is formedand isolating the formed exopolysaccharide wherein suitable growingconditions comprise growth in a so-called semi-defined medium consistingof a carbon source, at least one nitrogen source, a phosphate source insuch a concentration that it also has a high buffering capacity,vitamins, minerals, amino acids and peptide mixtures at a temperaturebetween 15° C. and 40° C.
 15. A process for in situ production of anexopolysaccharide according to claim 1 comprising growing aLactobacillus sake-like strain according to claim 14 in an edible mediumsuch as a dairy liquid under conditions whereby said EPS is formed untilthe number of Lactobacilli is in the order of 10⁷ -10¹¹ per ml.
 16. Aprocess for producing a food product containing a thickened aqueousphase, said process comprising incorporating into said food product aneffective amount of a polysaccharide according to claim
 1. 17. A processfor producing a mayonnaise type product in which milk or a milk-basedmedium is fermented with a Lactobacillus sake-like strain according toclaim 13 at 30° C. for 18 hours until a sufficient high amount ofLactobacilli is formed producing sufficient EPS followed by carefullymixing the fermented milk product with an aqueous phase containing usualingredients and with a separately prepared mayonnaise preemulsion phase.